The broad goals of this proposal are to understand the mechanisms underlying formation of the vertebrate primary mouth, using the frog Xenopus laevis as a model. The primary mouth (also called the stomodeum) is the first opening between the outside and the developing pharynx, and is the earliest element of craniofacial development. It is critical for food ingestion, and may contribute to the tongue and teeth. We previously defined multiple steps that occur during Xenopus primary mouth development, and isolated a set of regulatory genes whose expression is enriched in this region. An early, essential, step in primary mouth formation is local degradation of the basement membrane, which is mediated by local inhibition of the 2-catenin-mediated Wnt pathway. There are two Specific Aims. Aim 1 analyses regulation of basement membrane metabolism in the primary mouth region. Focus is on the role of matrix metalloproteases (MMPs) that degrade the basement membrane. Function of MMPs expressed in the developing primary mouth will be analyzed by antisense loss of function assays, and gain of function assays, where effects are localized using face transplants and temporally regulated promoter constructs. The hypothesis that basement membrane synthesis and degradation are balanced, and coordinately activated by Wnt signaling will be tested. Steps in primary mouth formation that are altered if the basement membrane does not break down will be defined. Aim 2 examines the signaling systems that direct primary mouth formation. One focus is on the kinin-kallikrein pathway that is required for primary mouth formation, but is poorly characterized during embryogenesis. The function during primary mouth formation of enzymes, receptors and modulators in this pathway will be tested by antisense loss of function and in vitro assays. Preliminary data will be extended to analyze the role of BMP and non-canonical Wnt signaling in the primary mouth region. Xenopus is an outstanding system for this project since tissue transplants can readily be used to localize effects of gene perturbation, whereas equivalent assays are difficult in mammalian models. Assays are rapid, as frog embryos develop rapidly; and frog genes are conserved with those of mammals. Steps occurring during formation of the mouse and chick primary mouth appear similar to those we have observed in Xenopus, indicating that our analyses will be useful for suggesting comparative studies in mammals. This study has broad significance. Craniofacial birth defects appear with high frequency, in 1/700 live births, however the etiology of most is unknown, and primary mouth perturbations have not generally been considered as causal. Both the interplay of Wnt and MMP signaling during basement membrane remodeling, and the kinin-kallikrein pathway are of likely significance in other embryonic organs. PUBLIC HEALTH RELEVANCE: This proposal analyzes formation of the primary mouth, the essential first opening from the outside of the embryo to the gut. We will identify and study the function of genes and that control primary mouth formation in the normal embryo. This study will suggest why the primary mouth fails to form correctly in a human embryo, and help explain the molecular causes of craniofacial birth defects.